The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including 18O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H2O2 concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data.

The low-molecular-mass fraction of the soybean nodule cytosol contains Fe capable of catalyzing free radical production through Fenton chemistry. A large portion of the pool of catalytic Fe, measured as bleomycin-detectable Fe, was characterized as complexes of Fe with phenolic compounds of three classes: phenolic acids, cinnamic acids, and flavonoids. Many of these compounds, along with other phenolics present in legume tissues, were used for a systematic structure-activity relationship study. All phenolics tested were able to chelate Fe, as judged from their inhibitory effect on site-specific deoxyribose degradation (minus EDTA assay). However, only those having catechol, pyrogallol, or 3-hydroxy-4-carbonyl groupings were potent chelators and reductants of Fe 3+ at pH 5.5.The same phenolics promoted oxidative damage to DNA (bleomycin assay) and to deoxyribose (plus EDTA assay), but inhibited linolenic acid peroxidation by chelating and reducing Fe 3+ and by neutralizing lipid radicals. Also, phenolics having a pyrogallol nucleus attenuated the free radical-mediated inactivation of glutamine synthetase, which was used as a model system, by chelating Fe 2+ . It is reasoned that under the microaerobic (10-20 nM O 2 ) and acidic (pH 5.5-6.4) conditions prevailing in nodules, phenolics are likely to act primarily as antioxidants, decreasing oxidative damage to biomolecules.

Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.

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Altogether, this study has important impacts on AIP care underlying that hemin needs to be restricted to severe neurovisceral crisis and suggests that alternative treatment targeting the liver such as ALAS1 and HO1 inhibitors, and anti-inflammatory therapies should be considered in patients with recurrent AIP.

Photoreceptor chromoproteins undergo light-induced conformational changes that result in a modulation of protein interaction and enzymatic activity. Bacterial phytochromes such as Cph1 from the cyanobacterium Synechocystis PCC 6803 are light-regulated histidine kinases in which the light signal is transferred from the N-terminal chromophore module to the C-terminal kinase module. In this study, purified recombinant Cph1 was subjected to limited proteolysis using trypsin and endoproteinase Glu-C (V8). Cleavage sites of chromopeptide fragments were determined by MALDI-TOF and micro-HPLC on-line with tandem mass spectrometry in an ion trap mass spectrometer. Trypsin produced three major chromopeptides, termed F1 (S56 to R520), F2 (T64 to R472), and F3 (L81 to R472). F1 was produced only in the far-red absorbing form Pfr within 15 min and remained stable up to >1 h; F2 and F3 were obtained in the red-light absorbing form Pr within ca. 5-10 min. When F1 was photoconverted to Pr in the presence of trypsin, this fragment degraded to F2 and F3 within 1-2 min. On size exclusion chromatography, F1 eluted as a dimer in the Pfr and as a monomer in the Pr form, whereas F2 and F3 behaved always as monomers, irrespective of the light conditions. These and other results are discussed in the context of light-dependent subunit interactions, in which amino acids 473-520 within the PHY domain are required for chromophore-module subunit interaction within the homodimer. V8 proteolysis yielded five major chromopeptides, F4 (T17 to N449), F5 (T17 to E335), F6 (T17 to E323), F7 (unknown sequence), and F8 (tentatively L121 to E323). F6 and F8 were formed in the Pr form, whereas F4, F5, and F7 were preferentially formed in the Pfr form. Three amino acids next to specific cleavage sites, R520, R472, and E323, were altered by site-directed mutagenesis. The mutants were analyzed by UV-vis spectroscopy, size exclusion chromatography, and autophosphorylation. Histidine kinase activity was low in R472A, R520P, and R520A; in all mutants, the ratio of phosphorylation intensity between Pr and Pfr was reduced. Thus, light regulation of autophosphorylation is negatively affected in all mutants. In R472P, E323P, and E323D, the phosphorylation intensity of the Pfr form exceeded that of the wild-type control. This result shows that the histidine kinase activity of Cph1 is actively inhibited by photoconversion into Pfr.

Systemic candidiasis remains a major cause of disease and death, particularly among patients suffering from hematological malignancies. In an attempt to contribute to the discovery of useful biomarkers for its diagnosis and therapeutic monitoring, we embarked on a mapping of Candida albicans immunogenic proteins specifically recognized by antibodies produced during the natural course of systemic Candida infection in this high-risk population. About 85 immunoreactive protein species were detected with systemic candidiasis patients' serum specimens by using immunoproteomics (i.e., two-dimensional electrophoresis followed by Western blotting), and identified through a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), de novo peptide sequencing using nano-electrospray ionization-ion trap (ESI-IT) MS, and genomic database searches. This proteomic approach has led to the characterization of 42 different housekeeping enzymes as C. albicans antigens. Their biological significance is also discussed. Furthermore, this study is the first to report that 26 of them exhibit antigenic properties in C. albicans, and 35 of them become targets of the human antibody response to systemic candidiasis. Our findings suggest that the production of antibodies to C. albicans phosphoglycerate kinase and alcohol dehydrogenase during systemic candidiasis could be associated with a differentiation of the human immune response. We also highlight the relationship between changes in maintenance of circulating levels of specific anti-Candida antibodies and patients' outcome. Some of these variations, especially the rise of high anti-enolase antibody concentrations, appear to be related to recovery from systemic candidiasis in these patients, which might serve as markers for predicting their outcome. This approach could therefore provide new challenges for diagnosis and clinical follow-up of these fungal infections, and even for antifungal drug or vaccine design.

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